建立了大鼠纹状体细胞膜色谱法研究5-羟色胺(5-hydroxytryptamine)受体1D亚型(5-HT1D)的别构效应.利用大鼠纹状体制备得到细胞膜色谱固定相,特异性地识别混合对照品中的尼群地平、欧前胡素和藁本内酯.以不同浓度(4.62× 10-7 ～ 4.62× 10-5 mol/L)的欧前胡素为竞争剂平衡细胞膜色谱柱,考察流动相浓度的变化对样品保留行为的影响.结果表明,欧前胡素的添加可使藁本内酯与受体作用的平衡解离常数(LD)增大2.06倍,说明欧前胡素结合5-HT1D受体后会影响藁本内酯在受体上的结合强度.在流动相中添加藁本内酯(5.26× 10-7 ～5.26×10-5 mol/L),考察欧前胡素保留因子的变化,测得藁本内酯的KD为(4.15±0.12) μmol/L,与文献报道值基本一致.该方法为定量研究受体的别构效应提供了一种全新思路.
A rat striatum cell membrane chromatography (CMC) method was developed for characterizing the allosteric effects for 5-hydroxytryptamine receptor subtype 1D (5-HT1D).The cell membrane stationary phase prepared by rat striatum was applied to specifically recognize the mixed standard solution of nitrendipine,imperatorin and ligustilide.Imperatorin was pumped through a CMC column continuously as a competitor to evaluate the effect of the competitor's concentrations on the retention of ligustilide.A decrease in the retention factor of ligustilide was observed in the range of 4.62× 10-7-4.62× 10-5 mol/L.The addition of imperatorin weakened the affinity that ligustilide binding to 5-HT1D by 2.06 times,which suggested that imperatorin occupied certain binding sites of 5-HT1D on the column thus hindering the association of ligustilide.For further competitive studies,the mobile phase containing ligustilide (5.26 ×10-7-5.26×10-5 mol/L) was pumped through the column,and imperatorin was injected onto the column.The equilibrium dissociation constant was (4.15± 0.12) μmol/L for ligustilide,which corresponded well with the previous studies.The results indicated that the CMC method could be a new way for quantitative analysis of allosteric effects for 5-hydroxytryptamine receptor.
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